Optineurin promotes myogenesis during muscle regeneration in mice by autophagic degradation of GSK3β

Skeletal muscle regeneration is essential for maintaining muscle function in injury and muscular disease. Myogenesis plays key roles in forming new myofibers during the process. Here, through bioinformatic screen for the potential regulators of myogenesis from 5 independent microarray datasets, we identify an overlapping differentially expressed gene (DEG) optineurin (OPTN). Optn knockdown (KD) delays muscle regeneration in mice and impairs C2C12 myoblast differentiation without affecting their proliferation. Conversely, Optn overexpression (OE) promotes myoblast differentiation. Mechanistically, OPTN increases nuclear levels of β-catenin and enhances the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription activity, suggesting activation of Wnt signaling pathway. The activation is accompanied by decreased protein levels of glycogen synthase kinase 3β (GSK3β), a negative regulator of the pathway. We further show that OPTN physically interacts with and targets GSK3β for autophagic degradation. Pharmacological inhibition of GSK3β rescues the impaired myogenesis induced by Optn KD during muscle regeneration and myoblast differentiation, corroborating that GSK3β is the downstream effector of OPTN-mediated myogenesis. Together, our study delineates the novel role of OPTN as a potential regulator of myogenesis and may open innovative therapeutic perspectives for muscle regeneration.


Introduction
Skeletal muscle, the most abundant tissue in our bodies, plays key roles in posture, mobility, and energy metabolism [1]. In muscle injury and muscular disease, the regeneration capacity of skeletal muscle is essential for restoration of these functions [2,3]. In response to muscle injury, satellite cells (SCs) will be activated to start myogenic differentiation, accompanied with up-regulated expression of myogenin (MYOG) and muscle-specific regulatory factor 4 [4,5]. The differentiation program is then completed with the activation of muscle specific proteins such as MYHC in myoblasts that subsequently fuse to regenerate myofibers to repair damaged muscle [2]. Therefore, myoblast differentiation-mediated myogenesis plays essential roles in muscle regeneration. Nonetheless, the underlying mechanisms of myogenesis during muscle regeneration remain largely unknown.
To explore the potential regulators of myogenesis, we performed bioinformatics screen from 5 myogenesis-related microarray datasets and identified optineurin (Optn) as one of the 5 overlapping genes, up-regulated during myoblast differentiation and muscle regeneration, while down-regulated in Duchenne muscular dystrophy (DMD) patients and mdx mice. The human OPTN is a 74-kDa scaffold protein comprised of 577 amino acids, and the mouse Optn gene encodes for a 584-amino acid protein (67 kDa), which is 78% identical to human OPTN [6]. OPTN is expressed in most tissues, including muscle, liver, and brain [7][8][9], and plays important roles in many cellular functions [6]. It has been identified as a selective autophagy receptor involved in the various stages of the autophagic process such as cargo recognition, autophagosome formation, and autophagic degradation [10]. OPTN mutations were shown in several familial diseases and often occur in its autophagy-associated ubiquitin-binding domain (UBAN) [11], such as OPTN E478G in amyotrophic lateral sclerosis (ALS) [7] and OPTN R545Q in normal-tension glaucoma [12]. OPTN is highly expressed in the skeletal muscle [13], yet little is known about the role of OPTN in myogenesis and whether its function in skeletal muscle is related to autophagy.
The canonical Wnt signaling pathway plays critical roles in facilitating the differentiation of SCs during skeletal muscle regeneration [14][15][16]. Wnt ligands bind to frizzled receptors and members of the low-density lipoprotein receptor related protein family, activating the nuclear translocation of β-catenin and the formation of a complex with the T-cell factor/lymphoid enhancer factor (TCF/LEF) [17]. It enhances transcriptional activity of myogenic factors such as myogenic factor 5 (Myf5) [18], myoblast determination protein (Myod) [19], fermitin family homolog 2 (Fermt2) [20], and Myog [21] during muscle regeneration. Wnts promote β-catenin nuclear translocation through inhibition of glycogen synthase kinase 3β (GSK3β), an important component of β-catenin destruction complex [22]. Genetic deletion or pharmacological inhibition of GSK3β leads to enhanced differentiation of C2C12 cells and muscle regeneration [23,24], indicating that GSK3β is essential for Wnt signaling pathway-mediated myoblast differentiation. Nevertheless, mechanism of Wnt-mediated suppression of GSK3β remains incompletely resolved and in dispute. A GSK3β-mediated crosstalk of autophagy and canonical Wnt signaling pathway has been shown during embryogenesis [25][26][27]. Whether the autophagy receptor OPTN is involved in the GSK3β-mediated canonical Wnt signaling pathway during muscle regeneration is completely unknown.
In this study, we show that OPTN is required for myoblast differentiation-mediated myogenesis during muscle regeneration in mice. OPTN promotes Wnt signaling pathway mediated myogenesis through direct physical interaction and autophagic degradation of GSK3β. Our findings reveal a new insight into mechanism underlying myogenesis during muscle regeneration and provide a potential target for muscle regeneration.

OPTN is essential for myogenesis during skeletal muscle regeneration
To investigate the role of OPTN in myogenesis during muscle regeneration, we analyzed OPTN expression during CTX-induced muscle injury in mice. Compared with uninjured muscle (day 0), OPTN expression was significantly up-regulated during the initial phase of muscle regeneration (day 5), and then decreased at day 14, similar to the expression profile of the newly regenerated myofiber marker embryonic myosin heavy chain (eMYHC) (Fig 1B,  S1A and S1B Fig). In line with this, the immunofluorescence analysis also showed increased OPTN in the cytoplasm of newly regenerated myofibers at 5 days postinjury ( Fig 1C, S2 Fig).
These results indicate a potential role of OPTN in muscle regeneration. We further generated a recombinant adeno-associated viral vector (AAV) with a short hairpin RNA (shRNA) targeting Optn (AAV-shOptn) (S3A and S3B Fig), which achieved 72% reduction in mRNA levels and 76% reduction in protein levels compared with scramble shRNA in tibialis anterior (TA) muscle (S3C and S3D Fig). The TA muscle receiving AAV scramble shRNA or shOptn was subjected to a single CTX injury and then allowed to recover for 3 to 14 days before analysis of the regenerated tissue. In scramble shRNA muscle, SCs descendants fused to form new myofibers characterized by centrally localized nucleus during the acute phase of regeneration (3 to 5 days after injury) (Fig 1D). The eMYHC + fibers were abundant at day 5 postinjury (Fig 1E). In contrast, AAV-shOptn muscle was composed of degenerating myofibers, fibrotic tissues, and inflammatory cells at this phase ( Fig 1D). The eMYHC + regenerating fibers in AAV-shOptn muscle were less and smaller at day 5 postinjury compared with scramble shRNA muscle ( Fig  1E-1G). In line with this, the protein levels of eMYHC and MYOG were significantly reduced in AAV-shOptn muscle at day 5 postinjury (Fig 1H-1J). Fourteen days after injury, muscle damage and inflammatory cells in scramble shRNA muscle were largely cleared, and the regenerated myofibers continued to grow and mature, as they became homogenous in size (Fig 1D), whereas small regenerated fibers and a few inflammatory cells were still shown in shOptn TA muscle ( Fig 1D). These data show that Optn knockdown (KD) delayed skeletal muscle regeneration in adult mice, indicating an essential role of OPTN in myogenesis during muscle regeneration.

OPTN promotes myoblast differentiation-mediated myogenesis
In response to muscle injury, muscle SCs undergo massive proliferation and differentiation to form new myotubes that replace the damaged myofibers [34]. To explore the role of OPTN in myogenesis during muscle regeneration, we first detected the effect of OPTN on muscle SCs proliferation and found similar paired box 7 + (Pax7) 5-ethynyl-20-deoxyuridine + (EdU) SCs frequency in shOptn and scramble shRNA TA muscle at day 3 postinjury (S4A-S4D Fig). In addition, Optn KD in C2C12 cells had no effect on numbers of EdU + cells and the expression of cell proliferation-associated genes (S4E-S4G Fig). These results suggest that OPTN does not affect SCs proliferation during muscle regeneration. We next investigated whether OPTNmediated myogenesis is achieved by regulation of myoblast differentiation. OPTN expression was increased during C2C12 myoblast differentiation with MYHC colocalization, similar to the expression patterns of MYOG (Fig 2A and 2B, S5A and S5B Fig). Optn KD in C2C12 cells

OPTN enhances myogenesis through activation of canonical Wnt signaling pathway
To explore the underlying mechanism of OPTN-mediated myogenesis, we analyzed the available gene expression profiles in Optn KD Hela cells (GSE6819), a common model for deep transcriptome analysis [35,36] and a fast way to gain preliminary indications. The result showed high implication of OPTN in the regulation of Wnt signaling pathway (Fig 3A), a wellcharacterized pathway in myogenesis [15], with down-regulation of Wnt signaling pathway target genes [MYC proto-oncogene (Myc) [37], cyclin D3 (Ccnd3) [38], twist family BHLH transcription factor 2 (Twist2) [39], and MYCN proto-oncogene (Mycn) [40]] ( Fig 3B). Consistent with this, we showed reduced mRNA levels of Wnts target genes in Optn KD C2C12 cells ( Fig 3C). TCF/LEF is the major transcription factor of Wnts target genes [41]. The TOP/ FOP reporter assay showed markedly decreased transcription activity of TCF/LEF in Optn KD C2C12 cells, while increased in Optn-OE C2C12 cells (Fig 3D). It has been shown that enhanced transcription activity of TCF/LEF is mediated by increased nuclear translocation of β-catenin, which further forms a complex with the TCF/LEF transcription factors to regulate Wnts target genes [17]. In agreement with this, Optn OE increased the nuclear levels of β-catenin in C2C12 cells, whereas Optn KD reduced these levels ( Fig 3E, S7A and S7B Fig). Similarly, Optn KD also decreased nuclear levels of active β-catenin in TA muscle at day 5 postinjury in mice ( Fig 3F). Furthermore, treatment of Wnt3a, a classical ligand of the Wnt signaling pathway [20], failed to effectively increase the nuclear levels of β-catenin in Optn KD C2C12 cells ( Fig 4A) and TA muscle at day 5 postinjury (Fig 4B), suggesting that OPTN is required for the activation of canonical Wnt signaling pathway. Together, these results suggest that OPTN activates canonical Wnt signaling pathway during myogenesis.

OPTN physically interacts with and targets GSK3β for autophagic degradation
Given that OPTN is a selective autophagy receptor [11], we deduced that OPTN might directly mediate the degradation of GSK3β. Cycloheximide (CHX) chasing assay showed approximately 7 hours half-life of GSK3β in si-control but approximately 10 hours in Optn KD cells Meanwhile, the ratio of LC3II toI, which correlates with autophagic flux and represents a reliable index of autophagy [46], was up-regulated in Optn OE C2C12 cells, while down-regulated in Optn KD C2C12 cells (Fig 5C and 5D, S8E Fig). These results indicate that OPTN degrades GSK3β through autophagy pathway. Immunoprecipitation assay revealed protein-protein interaction between OPTN and GSK3β in C2C12 cells ( Fig 5E). Immunofluorescence staining analysis showed colocalization of OPTN, GSK3β, and LC3 in C2C12 cells (S8F Fig). Optn KD reduced the interaction of LC3 and GSK3β in TA muscle at day 5 postinjury in mice as shown by immunoprecipitation (S8G Fig). Consistent with this, Optn KD decreased the colocalization of LC3 and GSK3β in C2C12 cells as shown by immunofluorescence staining (Fig 5F). Since OPTN has been shown to mediate autophagic degradation through LC3-interacting region (LIR) motif binding with LC3/GABARAP and UBANs binding with ubiquitin [47][48][49]  . Data are presented as mean ± SEM. � P < 0.05 versus control. The underlying data for this figure can be found in S1 Data. The original blot for this figure can be found in S1 Raw Image. AAV, adeno-associated viral vector; AMPK, AMP-activated protein kinase; Birc5, baculoviral IAP repeat containing 5; Ccnd3, cyclin D3; GEO, Gene Expression Omnibus; HIF, Hypoxia-( Fig 5G, S8I Fig). These data indicate that OPTN degrades GSK3β via activating LC3-mediated autophagy.

Inhibition of GSK3β rescues impaired myogenesis in Optn KD cells and skeletal muscle
To determine whether GSK3β is required for OPTN-mediated myogenesis, we inhibited the activity of GSK3β by its specific inhibitor CHIR and found rescued cell fusion and multinuclear myotube formation events in Optn KD cells (Fig 6A). The numbers of MyoG + cells were restored in Optn KD C2C12 cells treated with CHIR ( Fig 6B). Consistent with the morphological improvement, CHIR treatment recovered the down-regulated expression levels of MYHC and MYOG in Optn KD differentiating myoblasts (Fig 6C and 6D) and rescued the reduced nuclear levels of β-catenin in Optn KD C2C12 cells (Fig 6E and 6F). When CHIR was injected into the TA muscle treated with AAV-shOptn after CTX injury, the small size of eMYHC + inducible factor; H3, histone H3; KD, knockdown; KEGG, Kyoto Encyclopedia of Genes and Genomes; Myc, MYC proto-oncogene; Mycn, MYCN proto-oncogene; OE, overexpression; Optn, optineurin; PPAR, peroxisome proliferator-activated receptor; SEM, standard error of the mean; shRNA, short hairpin RNA; TA, tibialis anterior; Tcf7l2, transcription factor 7 like 2; TCF/LEF, T-cell factor/lymphoid enhancer factor; Twist2, twist family BHLH transcription factor 2.

Discussion
The regeneration ability of skeletal muscle has a high clinical relevance in some conditions such as postinjury recovery and muscular disease, and myogenic differentiation-mediated myogenesis is a pivotal step of muscle regeneration [2,51]. Here, we identified a selective autophagy receptor OPTN from 5 independent microarray datasets as a potential regulator of myogenesis. By performing a series of in vivo and in vitro experiments, we for the first time showed that OPTN activates Wnt signaling pathway mediated myogenesis through autophagic degradation of GSK3β during muscle regeneration. Our findings extend the understanding of regulatory mechanisms upon Wnt signaling pathway during muscle regeneration and reveal OPTN as a potential therapeutic target for defective muscle regeneration.
OPTN promotes myogenesis via activating canonical Wnt signaling pathway. The pathway is critical for myogenesis during muscle regeneration [52], regulating SCs differentiation through β-catenin binding with transcription factor 4 (TCF4) on the promoter of MYOG [53,54]. The stabilization of β-catenin is regulated by its destruction complex components. GSK3β, an important component of β-catenin destruction complex [45], is inhibited by Wnts to stabilize β-catenin and activate its nuclear translocation. There are several interpretations on Wnt-mediated inhibition of GSK3β in different models, including dissociation of GSK3β from AXIN via conformational changes [55] or posttranslational modifications [56], recruitment of GSK3 inhibitory proteins such as frequently rearranged in advanced T-cell lymphomas (FRATs) [57], or degradation of AXIN [58]. In addition, Wnts were reported to inhibit GSK3β activity by promoting its sequestration from the cytosol into multivesicular endosomes/bodies fusing with lysosome in 293T cells [59,60]. These findings shed fresh light on the inhibition of GSK3β and arouse broad interest in the field [61]. Based on the fact that the multivesicular endosome/body is an obligatory step before degradation in autophagosome [62,63], degradation might be the final fate of Wnt-induced GSK3β sequestration. Consistent with this, our data show GSK3β degradation mediated by OPTN in autophagosome during myogenesis, corroborating that OPTN involves in Wnt-mediated inhibition of GSK3β. These findings expand insights into the canonical Wnt/GSK3β/β-catenin signaling and reveal a novel mechanism on GSK3β inhibition in skeletal AU : TheabbreviationlistofFig7hasbeenupdated:Pleaseverifythata muscle.  OPTN is an autophagy receptor that plays a central role in selective autophagy [9]. Our data demonstrate that OPTN activates Wnt signaling pathway through autophagic degradation of GSK3β in muscle, suggesting an interaction between autophagy and Wnt signaling pathway during myogenesis. In fact, conflicting results on their relationship have been reported. Upregulation of autophagy is observed during the formation of mature myotubes and muscle regeneration [64][65][66], whereas inhibition of DVL2 autophagic degradation (a positive regulator of Wnt signaling pathway) is paradoxically capable of promoting Wnt signaling during muscle regeneration [67]. Similar results have been confirmed by Gao and colleagues, showing that autophagy inhibits Wnt signaling pathway by promoting Dvl2 degradation [68]. These results suggest negative regulation of Wnt signaling pathway by autophagy. Nevertheless, it has recently been reported that autophagy directly promotes nuclear translocation of β-catenin in a GSK3β dependent manner in C2C12 cells [69]. Consistently, our findings suggested that autophagy mediated OPTN-GSK3β axis exerts positive effects on Wnt signaling pathway during myogenic differentiation upon injury. In line with this, during hepatic progenitor cell differentiation, autophagy activates Wnt signaling pathway through interaction of p62 and phosphorylated GSK3β [70]. During adipocyte differentiation, a positive regulator of autophagy tumor protein P53 inducible nuclear protein 2 (TP53INP2) activates Wnt signaling pathway through autophagy-dependent sequestration of GSK3β [61]. Together, these findings suggest that the indeterminate intercommunication between autophagy and Wnt signaling pathway might be affected by dissimilar (negative or positive) regulators.

Fig 6. Inhibition of GSK3β rescues impaired myogenesis during myoblast differentiation induced by Optn KD. (A) Representative immunofluorescence staining of MYHC (left panel) and quantification of fusion index (a MYHC + cell with at least 3 nucleus) (right panel) in control (si-control) and Optn KD (si-Optn) C2C12 cells treated with CHIR (20 μM) or DMSO at 4 days postdifferentiation (n = 3 in each group). si-control or si-Optn were transfected into C2C12 cells for 48 hours before the initiation of differentiation. Scale bars: 20 μm. (B) Representative immunofluorescence staining of MYOG (left panel) and the percentage of MYOG + cells (right panel) in control (si-control) and Optn KD (si-Optn) C2C12 cells treated with CHIR or DMSO at 4 days postdifferentiation (n = 3 in each group
In summary, our data identify a novel function of OPTN for myogenesis during muscle regeneration. OPTN promotes myogenesis during muscle regeneration through autophagic degradation of Wnt signaling pathway inhibitor GSK3β (Fig 8). These findings uncover an OPTN/GSK3β/β-catenin axis that regulates myogenesis during muscle regeneration. Thus, OPTN may be a potential therapeutic target for the prevention and treatment of impaired myogenesis in injury and other muscular disease, such as DMD [71] and aging [72]. Further investigation is needed to assess the myogenic function of OPTN in advanced models to increase its impact on translational medicine.

Animal studies
Six-week-old male C57BL/6J mice purchased from the animal center of Xi'An Jiao Tong University (Xi'an, Shaanxi, PRC) were performed in accordance with the National Institutes of Health (Bethesda, Maryland, United States of America) GAU : Pleasenotethatasperstyle; italicsshouldno uide for the Care and Use of Laboratory Animals and with the approval of Animal Ethical and Welfare Committee of Northwest A&F University (Yang Ling, Shaanxi, PRC) [Approval ID: NWAFU-314031143]. All mice were housed with a 12-hour dark/light cycle with food and water ad libitum and were randomly allocated to the indicated groups. AAV serotype 9 vectors encoding a control scrambled shRNA sequence (scrambled; 5 0 -TTCTCCGAACGTGTCACGTAA-3 0 ) or a short hairpin targeting OPTN (shOptn; 5 0 -GCAAATGGCCATTCTTCTA-3 0 ) under the control of a U6 promoter and expressing EGFP (driven by a CMV promoter) were obtained from Hanbio (Shanghai, PRC). A single dose of 1.1 × 10 12 vg/mice in 40 μL of AAV2/9 expressing shOptn was delivered to 8-week-old mice injected locally into the right TA muscle, and the same dose of AAV2/9 expressing shRNA control was injected left TA muscle as AAV-shRNA control group. Production and purification of recombinant AAV were made by Hanbio. Mice were treated after recombinant AAV injection for 4 weeks. To induce muscle injury, 50 μl of 10 μM CTX (HF005, Heng Fei Biotechnology, Shanghai, PRC) was injected into TA muscle. The TA muscle was harvested at 0, 3, 5, and 14 days postinjury. To verify the activity of canonical Wnt signaling in vivo, the TA muscle of scrambled shRNA or shOptn was injected with 20 μl Wnt3a (100 ng/ml, 315-20, PeproTech, New Jersey, USA) or PBS (0.1% BSA) per mouse at 1.5 days postinjury [67]. The injured TA muscle was then collected for western blot analysis at 5 days postinjury. To inhibit the activity of GSK3β in vivo, the TA muscle of scrambled shRNA or shOptn was injected with 20 μl CHIR-99021 (50 ng/ml, HY-13259, MedChemExpress, Shanghai, PRC) or PBS (0.1% BSA) per mouse at 2.5 days postinjury [67]. The injured TA muscle was then collected for hematoxylin-eosin (HE) analysis and western blotting at 5 days postinjury.

Histological analysis
The TA muscle was fixed with 4% paraformaldehyde for more than 72 hours and then subjected to dehydration embedding. Finally, paraffin sections of muscle were obtained at a thickness of 2 to 4 μm for HE staining, and whole-slide digital images were collected with an Pannoramic DESK Scanner (P-MIDI, P250, 3D HISTECH, Hungary).

Plasmids and RNA interference
In order to construct a plasmid encoding HA-OPTN, HA-OPTN-F188A, and OPTN-E481G, the HA-tag was added at the N-terminus of OPTN, OPTN-F188A, and OPTN-E481G. The WT-OPTN, OPTN-F188A, and OPTN-E481G were cloned into the BamHI and XhoI sites of the pcDNA3.1-HA vector. In order to construct a plasmid encoding FLAG-GSK3β, the FLAG tag was added at the N-terminus of GSK3β. GSK3β was then cloned into the NotI and XbaI sites of the pcDNA3.1-FLAG vector. The pcDNA3.1-HA, pcDNA3.1-FLAG, and GFP-LC3B plasmid were provided by Dr. Qingzhu Sun (College of Animal Science and Technology, Northwest A&F University). The TOP flash/FOP flash and Renilla luciferase expression plasmids was provided by Dr. Qingyong Meng (College of Biological Sciences, China Agricultural University). The si-control, si-Optn, and si-β-catenin were synthesized from GenePharma (Shanghai, PRC). The sequences of Optn and β-catenin siRNAs were as follows: Optn siRNA1, 5 0 -GCAGACUUACCUGUUUCAATT-3 0 ; Optn siRNA2, 5 0 -GCAAAUGGCCAUUCUUCU ATT-3 0 .

Plasmid transfection and luciferase reporter assay
The plasmids were transfected into C2C12 cells and HEK293T cells using Lipofectamine 3000 (L3000001, Invitrogen, California, USA). In order to identify the activity of canonical Wnt signaling, the TOP flash/FOP flash expression plasmids with the Renilla luciferase expression plasmid were transfected when si-control and si-Optn C2C12 cells or HA-vector and HA-Optn were cultured after 2 days in the differentiation medium. The reporter activity was measured using the Dual-luciferase Reporter Assay System (E1980, Promega, Wisconsin, USA).

Immunofluorescence
Muscle sections and cultured cells were fixed in 4% formaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes on ice, and then blocked in 3% bovine serum albumin in PBS for 1 hour. The samples were blocked in 5% BSA for 2 hours at room temperature. Primary antibodies listed in S1 Table were incubated in blocking buffer at 4˚C overnight. Subsequently, the samples were washed with PBS and stained with the appropriate fluorescently labeled secondary antibodies (fluorescein isothiocyanate or rhodamine) for 1 hour at room temperature. After washing with PBS, DAPI (C0060, Solarbio, Beijing, PRC) was used to stain nucleus for 3 minutes. For immunostaining of muscle sections, whole-slide digital images were collected with an Pannoramic DESK Scanner (P-MIDI, P250, 3D HISTECH). Cross-sectional area of the new myofibers was calculated on section images obtained from TA muscle using ImageJ. For immunostaining of cultured cells, images were acquired using BioTEK gen 5 Software. Total cell nucleus and nucleus within myotubes were counted using ImageJ. The distribution of nucleuses per myotube and fusion index (a MYHC + cell with at least 3 nucleuses) was calculated as the number of nucleuses in myotubes divided by the total number of nucleuses counted.

Real-time reverse transcriptase PCR
Real-time PCR were performed as described [73]. Total RNA was isolated from the fresh TA muscle using TRIzol reagent (9109, Takara, Shiga, Japan). Complementary DNA (cDNA) was synthesized from total RNA using cDNA synthesis kit (R333-01, Vazyme Biotech, Nanjing, China) following the manufacturer's instructions. RT-PCR was performed using a CFX 96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). Each 20 mL amplifications contained 10 μl of ChamQ SYBR qPCR Master Mix (Q222-01, Vazyme Biotech), 7.8 mL of sterilized double-distilled water, 1 mL of 1:10 diluted cDNA, and 0.6 mL of each forward and reverse primer. The RT-qPCR program comprised an initial activation step at 95˚C for 3 minutes, followed by 38 cycles of 95˚C for 15 seconds and 60˚C for 30 seconds, and 5 seconds at 65˚C. After the PCR, a single product generated in these reactions was confirmed via melting curve analyses. The comparative Ct method (2 −ΔΔCt ), described in the literature [74] was used to calculate the gene expression values. The primer sequences for genes were listed in S2 Table. Immunoblotting C2C12 cells and TA muscle were washed with PBS and lysed in RIPA lysis buffer (P0013C, Beyotime Biotechnology, Shanghai, PRC). Next, 200 μg of total protein was resolved by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) (IPVH00010, Millipore, Massachusetts, USA) membrane via electroblotting. The PVDF membrane was blocked in black buffer (5% skim milk powder dissolved in TBST) for 2 hours at room temperature. Primary antibodies listed in S1 Table were applied in TBST at 4˚C overnight. Subsequently, the PVDF membrane was washed 4 times with TBST (5 minutes per time) and stained with the secondary antibodies (goat anti-rabbit or mouse) for 2 hours at room temperature. After washing with TBST, the ECL Reagent (WBKlS0100, Millipore) was used, and the strips were on film.

Immunoprecipitation
For immunoprecipitation analysis, the TA muscle and cultured cells were homogenized with IP lysis buffer (containing 1M pH 7.4 Tris-HCl 25ml, NP40 25ml, NaCl 4.383g, EDTA 0.146g, glycerin 50 ml, and protease inhibitor cocktail), and the total protein was incubated with 5 μg of the Rabbit monoclonal antibody to HA, GSK3β, or nonspecific Rabbit IgG for 2 hours at room temperature and then immunoprecipitation with protein A/G magnetic beads (B23201, Bimaker, Shanghai, PRC) at 4˚C overnight. After washing 3 times with TPBS (5 minutes per time), the protein-bound beads were finally resuspended in 20 μl 1× SDS-PAGE loading buffer. The samples were boiled at 95˚C for 10 minutes, and the supernatant was loaded on the gel for immunoblotting.

Treatment with reagents in cell culture
In order to verify the activity of canonical Wnt signaling, si-control and si-OPTN C2C12 cells were treated with either Wnt3a (100 ng/ml, 315-20, PeproTech) or PBS for 24 hours after 2 days in differentiation medium. The cell samples were then collected for western blot analysis. In order to inhibit the activity of GSK3β in OPTN KD C2C12 cells, the OPTN KD C2C12 cells were treated with CHIR-99021 (CHIR; 3 μM, HY-10182, MedChemExpress) during differentiation. Western blotting and immunofluorescence analysis were performed after 4 days of differentiation. For CHX chasing assay, cells were treated with CHX (50 μg/ml) and collected at the indicated time points and prepared for western blot analysis. To determine which degradation system dominantly controls the degradation of GSK3β, DMSO, MG132 (25 μM, HY-13259, MedChemExpress), and 3-MA (5 mM, HY-19312, MedChemExpress) were added to C2C12 cells cultured for 6 hours with or without Optn OE to detect the protein expression of GSK3β via western blotting.

Statistical analysis
All experiments were at least performed in 3 independent experiments. Data are presented as mean ± standard error of the mean and were analyzed by 2-tailed Student t tests for comparisons between 2 groups or 2-way analysis of variance (ANOVA) with Duncan post hoc test for multiple comparisons. Statistical significance was defined as � P < 0.05 versus controls. All data were analyzed using PASW Statistics 20 (SPSS, Chicago, Illinois, USA). . (E) Representative EdU and DAPI staining analysis in control (si-control) and Optn KD (si-Optn) C2C12 cells. si-control or si-Optn were transfected into C2C12 cells for 24 hours before staining analysis. Scale bar: 300 μm. (F) Quantification of the percentage of EdU-positive cells/total cells in control (si-control) and Optn KD (si-Optn) C2C12 cells (n = 5 in each group). si-control or si-Optn were transfected into C2C12 cells for 24 hours before staining analysis. (G) Representative mRNA expression analysis of cell proliferationassociated genes in C2C12 cells with si-control or si-Optn transfection (n = 6 in each group). Cells were collected after 24h transfection. Data are presented as mean ± SEM. � P < 0.05 versus control. The underlying data for this figure can be found in S1 Data. AAV, adeno-associated viral vector; EdU, 5-Ethynyl-2 0 -deoxyuridine; KD, knockdown; OPTN, optineurin; Pax7, paired box 7; SEM, standard error of the mean; shRNA, short hairpin RNA; TA, tibialis anterior. (TIF)

S5 Fig. The quantification of OPTN and MYOG immunoblotting analysis in C2C12 cells during differentiation. (A)
The quantification of OPTN immunoblotting analysis in C2C12 cells during differentiation at the indicated time points (0, 2, 4, 6, and 8 days) (n = 3 in each group). (B) The quantification of MYOG immunoblotting analysis in C2C12 cells during differentiation at the indicated time points (0, 2, 4, 6, and 8 days) (n = 3 in each group). Data are presented as mean ± SEM. � P < 0.05 versus control. The underlying data for this figure can be found in S1 Data. MYOG, myogenin; Optn, optineurin; SEM, standard error of the mean. (TIF) S6 Fig. OPTN promotes myoblast differentiation mediated myogenesis. (A, B) Representative immunoblotting analysis (A) and quantification (B) of OPTN, MYHC, and MYOG in sicontrol or si-Optn C2C12 cells at 4 days postdifferentiation (n = 3 in each group). si-control or si-Optn were transfected into C2C12 cells for 48 hours before the initiation of differentiation.